rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

Watarai M, Kamata Y, Kozaki S, Sasakawa C - J. Exp. Med. (1997)

Bottom Line:
Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin.Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA.Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase.

Affiliation: Department of Bacteriology, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.

ABSTRACTShigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

Mentions:
The secretion of Ipa invasins from the surface of S. flexneri into the external medium is essential for allowing the bacteria to invade epithelial cells (14, 35). We therefore wished to clarify that the CHO cell response to YSH6000T invasion observed in this study was the consequence of the activity displayed by the Ipa invasins secreted into the medium. To test this, semiconfluent CHO cell monolayers serum starved for 3 d were incubated in tissue culture medium (MEM) containing secreted IpaB, IpaC, and IpaD and examined for the appearance of actin polymerization, foci of protein tyrosine phosphorylation, and accumulation of vinculin and talin. As shown in Fig. 7 A, a dramatic change in the development of actin polymerization in CHO cells incubated in Ipa-containing MEM was accompanied by the appearance of foci of protein tyrosine phosphorylation around the peripheral edges. Similar responses were also observed in Swiss3T3 fibroblasts (Fig. 7 B). As shown in Fig. 7 C, the semiquantitative measurement of amounts of actin polymerization, and accumulation of vinculin, talin, paxillin, FAK, and tyrosine phosphorylated proteins in the CHO cells revealed that at 30 (lane 2) and 60 min (lane 3) after the incubation was increased to greatly higher levels as compared with those of the CHO cells incubated in the MEM at 0 min (lane 1). Such cellular responses were not evoked at 60 min incubation in the CHO cells incubated in the MEM whose Ipa invasins had been removed by immunoprecipitation with anti-IpaB, -IpaC, and -IpaD antibodies (lane 4). The cellular responses to the addition of Ipa invasins were almost completely shut off in CHO cells pretreated with C3 (lane 5). In fact, measurement of phosphorylated FAK and paxillin levels by immunoblottings with anti-phosphotyrosine revealed that they were elicited upon incubation of the CHO cells in Ipa-containing MEM 30 min after incubation (Fig. 7 D). These data thus indicate that the cellular responses to the addition of secreted Ipa invasins are regulated by rho.

Mentions:
The secretion of Ipa invasins from the surface of S. flexneri into the external medium is essential for allowing the bacteria to invade epithelial cells (14, 35). We therefore wished to clarify that the CHO cell response to YSH6000T invasion observed in this study was the consequence of the activity displayed by the Ipa invasins secreted into the medium. To test this, semiconfluent CHO cell monolayers serum starved for 3 d were incubated in tissue culture medium (MEM) containing secreted IpaB, IpaC, and IpaD and examined for the appearance of actin polymerization, foci of protein tyrosine phosphorylation, and accumulation of vinculin and talin. As shown in Fig. 7 A, a dramatic change in the development of actin polymerization in CHO cells incubated in Ipa-containing MEM was accompanied by the appearance of foci of protein tyrosine phosphorylation around the peripheral edges. Similar responses were also observed in Swiss3T3 fibroblasts (Fig. 7 B). As shown in Fig. 7 C, the semiquantitative measurement of amounts of actin polymerization, and accumulation of vinculin, talin, paxillin, FAK, and tyrosine phosphorylated proteins in the CHO cells revealed that at 30 (lane 2) and 60 min (lane 3) after the incubation was increased to greatly higher levels as compared with those of the CHO cells incubated in the MEM at 0 min (lane 1). Such cellular responses were not evoked at 60 min incubation in the CHO cells incubated in the MEM whose Ipa invasins had been removed by immunoprecipitation with anti-IpaB, -IpaC, and -IpaD antibodies (lane 4). The cellular responses to the addition of Ipa invasins were almost completely shut off in CHO cells pretreated with C3 (lane 5). In fact, measurement of phosphorylated FAK and paxillin levels by immunoblottings with anti-phosphotyrosine revealed that they were elicited upon incubation of the CHO cells in Ipa-containing MEM 30 min after incubation (Fig. 7 D). These data thus indicate that the cellular responses to the addition of secreted Ipa invasins are regulated by rho.

Bottom Line:
Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin.Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA.Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase.

Affiliation:
Department of Bacteriology, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.

ABSTRACTShigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.